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Partial Characterization of the Potential Biodegrading Ability of Xylaria sp. on Natural
Rubber, Chicken Feathers, and Polystyrene
A Thesis Proposal
In Partial Fulfillment of the
Requirements in
Biology 200: Undergraduate Thesis
AY 2008-2009
Dayao, Janine Erica P.
Egloso, Mary Bernadette V.
August 15, 2008
INTRODUCTION
Background of the Study
Pollution is an inevitable problem due to population growth, urbanization and the
increased demand for manufactured products in the local and export markets.
Industrialization has resulted to the generation of wastes of various forms that pose serious
risks to the environment and public health, thus, requiring an efficient waste regulatory
management.
At the advent of technology, pollution has indeed taken its toll on nature, making
people harvest and manufacture products that would take eons to decay and rot at the very
least. Everywhere, a plethora of biodegradable and non-biodegradable wastes can be
seen. And, indeed it’s high time that people revert back to natural processes that could help
solve the burgeoning problem of waste disposal since all the artificial methods that require
today’s technology could contribute to the pollution that the planet’s experiencing now. One
such natural process that could solve the problem, or in a way even just alleviate such
waste pile-up, is biodegradation.
The country’s population growth rate is one of the highest in the world (Mangahas,
2006) and it places serious strains on the economy. In 2005, the population was 82.8
million, of which 51.8 million or 63% lived in urban areas. Metro Manila is the most densely
populated urban area with 10.7 million (Mangahas, 2006). Over the past 3 decades, the
country’s economy slid behind many Asian economies. Gross domestic product (GDP)
grew at an average of only 3%, compared with 8% in the People’s Republic of China
(PRC); 6% in the Republic of Korea, Singapore, Malaysia, and Thailand; and 5% in
Indonesia over the last 30 years (Wallace Report 2004 as cited by Mangahas, 2006).
Urbanization, decline in the economy and further population growth lead to the even higher
generation of wastes that has not been managed properly and safely (DENR, 2004).
Presidential Decree (PD) 1152, or “the Philippine Environmental Code,” provides the
basis for an integrated waste management regulation starting from waste source to
methods of disposal. PD1152 has further mandated specific guidelines to manage
municipal wastes (solid and liquid), sanitary landfills and incineration, and disposal sites in
the Philippines. Apart from the basic policies of PD1152, waste management must also
comply with the requirements and implementing regulations of other specific environmental
laws, such as PD984 (Pollution Control Law), PD1586 (Environmental Impact Assessment
System Law), RA8749 (Clean Air Act) and RA9003 (Ecological Solid Waste Management
Act) (DENR, 2004).
A study by Clutario and Cuevas (2001) showed that Xylaria sp. can utilize
polyethylene plastic strips as an alternative carbon source. The fungus grew optimally at
250C on a mineral medium of pH5 containing 0.5% glucose and polyethylene plastic strips
as co-carbon source. A mucilaginous sheath was produced by the fungus to help its
mycelial growth adhere to the surfaces and edges of the plastic strips. After 50 days of
incubation, the strips became embedded in the mycelial growth. Visible damage on the
surface structure of the plastic strips was observed using scanning electron microscopy
(SEM). Striations and tearing were present due to the active burrowing of Xylaria hyphae
on the polyethylene material. This shows that Xylaria sp. has indeed a potential in
degrading synthetic wastes like plastics which are difficult to decompose.
This proposed study aims to test the potential use of Xylaria sp. and its mutants as a
natural biodegrading agent in biodegrading other rampant wastes such as natural rubber,
polystyrene and chicken feathers.
Objectives
The proposed study aims to determine the biodegrading capacity of Xylaria sp. wild
type and its mutants.
The specific objectives are as follows:
1. to determine if Xylaria sp. mutants and wild type can degrade natural rubber as a
carbon source
2. to determine if Xylaria sp. mutants and wild type can degrade chicken feathers as a
carbon and nitrogen source
3. to determine if Xylaria sp. mutants and wild type can degrade/consume/ assimilate
polystyrene as a carbon source
4. to compare the biodegrading ability of the wild type to each mutant to find which
strain is most appropriate for each type of waste
5. to examine the treated pollutants under the scanning electron microscope (SEM) to
check if the pollutants have been biodegraded by the Xylaria sp. strains
Significance of the Study
Findings of this study might be utilized in the development of Xylaria sp. as a good
biodegrading agent in reducing durable wastes such as plastics and others, as well as in
optimizing fungal technologies. This study may also provide a way or ways in the discovery
of other important characteristics of Xylaria sp. and its mutants, which may be used in other
applications and scientific investigations. The discovery of other sources of biodegradation
agents and their potential bioactive natural products is of paramount importance, especially
nowadays that people should mostly concern about their waste disposal methods, and also
to assure a good source of more accessible ways, through research, in approaching the
reduction of pollution that are safe and can possibly boost the Philippine fungal industry in
the world market.
Scope and Limitations
The experiment will serve as a source of preliminary information on the potential of
Xylaria sp. strains to degrade chicken feathers, polystyrene and natural rubber. Other
pollutants with similar biochemical structure to the aforementioned pollutants will not be
included in the experiment. For the methodology, the Xylaria sp. that will be used will only
come from the stock culture of UP Los Baños Biotech Institute. Culture media and reagents
will also be provided by BIOTECH. Experimentation will be done both in the Microbiology
thesis room of the UP Manila, College of Arts and Sciences and in the Antibiotic Laboratory
in BIOTECH. Isolation and purification of active components (i.e. enzymes) responsible for
the probable degradation of chicken feathers, polystyrene and natural rubber will not be
performed. Observation of colonization through scanning electron microscopy (SEM) will be
done in BIOTECH. Only the crude weight percent difference of the colonization of the
wastes will be recorded. The runs will only be done twice due to logistic matters such as
paucity and unavailability of equipment. Moreover, observations of the set-ups will be noted
on the 20th, 30th and 50th day of incubation, with 50 days as the maximum incubation
period. In terms of data analysis, this experiment will only focus in analyzing the
degradation potential of Xylaria sp. strains on natural rubber, chicken feathers and
polystyrene. Also, it will be concerned on whether the degradation capacity of the mutant
strains, which will be described later, is significantly different from the capacity of the wild
type to degrade.
REVIEW OF RELATED LITERATURE
BIODEGRADATION
Biodegradation is the breakdown of natural substances through the action of
enzymes secreted by organisms such as microbes and fungi. Only waste materials made
up of natural polymers can be degraded by microbes and fungi. Biodegradation works in
such a manner that the organisms involved utilize, or more appropriately, metabolize these
wastes as sources of nutrients such as carbon or nitrogen. (Tortora, et al., 2005).
In many cases, conditions are not favorable enough to promote spontaneous
biodegradation or natural attenuation. Whenever there is insufficient quantity of naturally
biodegrading organisms, there is a further need to add nutrients or other suitable
organisms aside from those present already, for improved biodegradation to occur. The
general objective of biodegradation is to discern the speed of unaided biodegradation
before catalysts may even be added, and then strengthen spontaneous biodegradation
only if this is not fast enough to remove the contaminant’s concentration in the environment
at which it may cause health risks to nearby inhabitants such as people, animals and plants
(European Federation of Biotechnology, 1999).
MICROORGANISMS USED IN BIODEGRADATION
Through time, scientific experiments have already proven the ability of some
microorganisms to biodegrade pollutants such as polyethylene, polystyrene, rubber,
chicken feathers and other types of wastes. Organisms such as bacteria and fungi have
proven themselves to possess the capacity to biodegrade pollutants.
Bacteria such as Brevibaccillus borstelensis, Rhodococcous rubber C208,
Xanthomonas sp. strain 357 have been proven to degrade pollutants. Plastics in the form of
polyethylene are known to be degraded by the thermophilic bacterium Brevibaccillus
borstelensis 707 which was isolated from soil. The biodegradation was described through
weight reduction by 11-30%. And the incubation lasted for 30 days. (Hadad et al, 2005).
Another study by Orr et al. (2004) featured the Rhodococcous ruber C208 as an effective
polyethylene-degrading organism. In addition to this, this strain has been proven to
degrade polystyrene (Mor and Sivan, 2008). Yet originally, Rhodococcus rubber is a known
rubber-degrading organism, according to the review of Rose and Steinbuchel (2005).
Xanthomonas sp. strain 357, in much the same way, can degrade rubber too.
A number of fungi species are also known to biodegrade. The known fungi
biodegraders are Gordonia sp., Streptomyces sp., Nectria gliocladioides, Penicillium
ochrochloron and Geomyces pannorum and Trichoderma atroviride. (Barraat et al.,2003;
Cheng chang et al., 2003; Rose and Steinbuchel, 2005)
Gordonia sp. and Streptomyces sp. are known rubber-degraders (Rose and
Steinbuchel, 2005). Nectria gliocladioides (five strains), Penicillium ochrochloron (one
strain) and Geomyces pannorum (seven strains), in a study of Barraat et al. (2003), have
been observed to degrade polyurethane while simultaneously relating it to the water
holding capacity of the soil. Moreover, In a study conducted by Cao et al. (2008), the
fungus Trichoderma atroviride completely degraded the chicken feathers. This strain was
actually isolated from a decaying feather.
The list of microorganisms that could be used in biodegrdation goes on for there are
still more species that could degrade pollutants. And in fact, in the Philippines a fungus has
been isolated and proven to degrade polyethylene (Cuevas and Manaligod, 1997).
Xylaria sp. AS A POTENTIAL AGENT FOR BIODEGRADATION
Xylaria sp. was discovered by Cuevas and Manaligod (1997), growing on a sando
plastic bag, buried in forest soil and litter in the lowland secondary forest of Mt. Makiling,
Laguna. The fungus comprised of sterile melanin pigmented mycelia and was reported as
ascomycete sterile dark mycelia (ASDM). Cultural studies have designated it under Class
Ascomycetes, Order Xylariales, Genus Xylaria (Clutario and Cuevas, 2001).
A previous study by Clutario and Cuevas (2001) proved that Xylaria sp. can utilize
polyethylene plastic strips as an alternative carbon source, thereby degrading them into
usable forms for self-sustenance. Through the use of scanning electron microscopy, the
proponents of the said study observed visible damages of the surface structure of the
plastic strips. There were tearing and striations caused by active burrowing of Xylaria
hyphae on the polyethylene material. Plastic is an extremely versatile synthetic material
made of high molecular weight, semi-crystalline polymer prepared from ethylene through
the cracking of crude oil, light petroleum and natural gas. For plastic bags alone, it is
estimated that some 430,000 gallons of oil are needed to produce 100 million pieces of
these omnipresent consumer items on the planet (Knapczyk and Simon, 1992; EcoWaste
Coalition, 2008).
Figure 1. Xylaria in its natural habitat.
Figure 2. Xylaria fruiting body surrounded by flask-shaped structures called peritheca.
Figure 3. Cross section of Xylaria stroma.
Xylaria is one of the most commonly encountered groups of ascomycetes with most
of its members being stromatic, peritheciate, with an iodine-positive ascus apical ring, and
with one-celled, dark ascospores on which agermination slit can be found. Most are
inhabitants of wood, seeds, fruits, or leaves of angiosperms. Some are associated with
insect nests. Most decay wood and many are plant pathogens. Many are endophytes. They
are commonly found throughout the temperate and tropical regions of the world. The
Xylaria sp. can be distributed above, around, and beneath perithecia. It forms a unipartite
stromatal layer, with a superficial or erumpent surface level. The interior of its stromata is
essentially homogeneous. Conidium-bearing discs, potassium hydroxide pigments and
orange granules surrounding the perithecia are absent (Rogers et al., 2002). They are
mostly multiperitheciate in ascomatal number per stroma, ascomatal ostioles and ascal
apical rings: are present, and the ascospore cell number is one-celled. Teleomorph and
anamorph are produced on the same stromata in most species, with their anamorphs:
Geniculosporium-like.
Some Xylaria sp. species exist as endophytes, and have mutualistic associations
with plants. The fungus secrete toxins to protect the plant from herbivory from other insects
or animals, while the fungus in return feeds on the host’s tissues for nutrition, and its
mycelia are scattered through seed dispersal. Xylariaceae endophytes are hypothesized to
be quiescent colonizers that decompose lignin and cellulose when a plant dies.
Nonetheless there are also some xylariaceous fungi that only exist as endophytes. No
obvious benefit to living host plants has been documented for Xylariaceae (Petrini et al.,
1995; Whalley, 1996; Rogers, 2000; Davis, et al., 2003)
A review of empirical studies on antagonistic interactions between endophytes and
grazers, insects and microbial pathogens summarizes five general properties of endophyte
mutualism: (1) the endophyte is ubiquitous in a given host, geographically widespread, and
causes minimal disease symptoms in the host plant; (2) vertical transmission or efficient
horizontal transmission of the fungus occurs; (3) the fungus grows throughout host tissue,
or, if confined to a particular organ, a high proportion of such organs are infected; (4) the
fungus produces secondary metabolites likely to be antibiotic or toxic; and (5) the
endophyte is taxonomically related to known herbivore or pathogen antagonists (Carroll,
1988; Davis, et al., 2003).
SOLID WASTE IN THE PHILIPPINES
Filipinos generate around 0.3 to 0.7 kilograms of garbage daily per person
depending on income levels (World Bank, 2001). Metro Manila produces about 8,000 tons
of solid waste each day and is expected to reach 13,300 tons each day in 2014 (Baroña,
2004). The National Capital Region produces the highest amount of wastes, about 23% of
the country’s waste generation (Anden and Rebolledo, 2003).
Based on studies (2001) made by the National Solid Waste Management
Commission Secretariat based at the Environmental Management Bureau (EMB), it is
estimated that in Metro Manila, the per capita waste production daily is 0.5 kg. Thus, every
person living in the metropolis generates half a kilo of waste a day. With an estimated
population of 10.5 million, total waste generated in Metro Manila alone could run up to
5,250 metric tons per day or 162,750 metric tons per month or 1.95 million metric tons per
year.
Based on another EMB study (2001) regarding the disposal of daily wastes, only
about 73% of the 5,250 metric tons of waste generated daily are collected by dump trucks
hired by local government units. The remaining 27% of daily wastes, or about 1,417.5
metric tons, end up in canals, vacant spaces, street corners, market places, rivers and
other places.
According to a survey conducted by the EcoWaste Coalition and Greenpeace
Southeast Asia in 2006, synthetic plastics comprise 76% of the floating trash in Manila Bay,
out of which 51% are plastic bags, 19% are sachets and junk food wrappers, 5% are
styrofoams and 1% is hard plastics. The rest were rubber (10%) and biodegradable
discards (13%) (EcoWaste Coalition, 2008).
Polystyrene
Polystyrene, an aromatic polymer, is synthesized from the aromatic monomer
styrene which comes from petroleum products. It is a thermoplastic substance that could be
solid in room temperature or liquid when melted. One of the most common forms and uses
of polystyrene is the EPS which stands for Expanded Polystyrene. The industry
manufactures such product by mixing polystyrene with blowing agents in the form of carbon
dioxide and pentane which comprises 5%-10% of its composition. The EPS is also called
foamed polystyrene and it is said to be 30 times lighter than regular polystyrene. This
substance is popularly used in the form of beverage cups and insulating materials. (Friend,
2005). The basic unit of polystyrene which is styrene, which is a known neurotoxin and
animal carcinogen, is considered very harmful to human health. In fact, it inflicts
neurological and hematological disorder especially to factory workers. EPS food packaging
is the one accountable for the leaking out of styrene. Styrene leak or leech is triggered
when acids from our juices when placed in such EPS cups and when food with Vitamin A
content is placed inside a microwave leading the styrene to accumulate in our system.
(Californians Against Waste, 2008).
Polystyrene is in high demand. It is the most used and utilized thermoplastic in the
industry due to its durability. But it is not biodegradable. (Mor and Sivan, 2008). According
to the Californians Against Waste (2008), it is very difficult to recycle due to its light weight
property, which accounts for why it’s expensive to recycle. Imagine just recycling a ton of
polystyrene, needs a budget of $3000. Hence, it has a negative scrap-value. More so, it’s
due to this light weight property that they find polystyrene hard to transport since
polystyrene is advised to be always kept food-free and uncontaminated when recycled. The
build-up of polystyrene in landfills, as reported by CAW (2008), will contribute to plastic
marine debris, since even when it is disposed of properly it is carried by natural agents
such as wind or other forces to the ocean. As manifested, there is an excess of it in the
environment and it is a major pollutant. (Mor and Sivan, 2008). For almost three decades
ago, polystyrene was first ban due to the utilization CFC material for its generation. In fact
there was a hype heralding that it is recyclable. After some time the companies that
invested for its recycling process disappeared. This move confirms that, indeed, recycling
polystyrene is not an easy thing to do. Now, the problem is back and the attention of
scientists is focused on the recycling of disposable foamed polystyrene. But recycling it
would cost much in terms of energy, waste and management point of view. (Californians
Against Waste, 2008). A way of solving such impending problem, is through biodegradation
(Mor and Sivan, 2008; Singh and Sharma, 2007).
Biodegradation has been manifested in a number of studies already. And some of
the studies will be named here. A study by Mor and Sivan (2008), dealt with the monitoring
of biofilm formation of the microbe Rhodococcus sp. strain C208 on polystyrene. Their aim
was to observe the kinetics of biofilm formation and of whether polystyrene would be
degraded. They used two methods in quantifying the biofilm biomas: modified crystal violet
staining and observation of the protein content of the biofilm. The C208 strain was cultured
in a flask containing polystyrene flakes with the addition of mineral oil (0.0055% w/v), which
induced more biofilm buil-up. The study concluded that after an extension of 8th weeks of
incubation, loss of 0.8% (gravimetric weight loss) of polystyrene weight was found. From
this, Mor and Sivan (2008) regarded C208 to demonstrate a high affinity towards
polystyrene through biofilm formation which lead to it’s degradation. The C208 strain is a
biofilm-producing actinomycete that has first colonized and degraded polyethylene (Orr et
al., 2004).
There were studies that tested the possibility of whether copolymerizing polystyrene
with other substance could make it more degradable and susceptible to microbial attack. In
1992, a study by Milstein, et al. (1992), focused on the biodegradation of a lignin-
polystyrene copolymer. The white rot basidiomycete was used to degrade such lignin-
polystyrene complex copolymer. Such fungi released enzyme that oxidized lignin and
demonstrated the degradation through weight loss, UV spectrophotometric analysis and
deterioration of surface of the plastic substance as seen under the SEM. A similar study by
Singh and Sharma (2007) demonstrated through the process of graft copolymerization that
polystyrene must be modified with natural polymers and hydrophilic monomers so as to
enhance its degrading ability and so as to render polystyrene waste useful in diminishing
metal ion pollution in water and. According to the mentioned study, the degrading rate of
polystyrene increased to 37% after subjecting it to soil burial method for 160 days.
Furthermore, the study of Motta et al. (2007), explored the degradation of oxidized
polystyrene using the fungi Curvularia sp. After about nine weeks of incubation,
microscopic examination revealed that hyphae had grown on the polystyrene. The
colonization of the fungi and it’s adhesion to the surface of the substance, according to
Motta, et al. (2007), is a crucial step towards polymer biodegradation.
As demonstrated, colonization is needed in determining whether a particular microbe
or organism is a potential biodegrading agent. (Motta et al., 2007) The growth of the
microbes on the surface of the polystyrene is a step that would lead to its degradation.
Further visual confirmation of deterioration of surface area is done by using the scanning
electron microscope. (Mor and Sivan, 2008; Motta et al., 2007).
Natural Rubber
Natural rubber (NR) is made from the latex of the Hevea brasiliensis also known as
the rubber tree. It is mainly composed of cis-1,4 polyisoprene which has a molecular mass
of about 106 Da. This could also be chemically synthesized and produce the substance
known as Isoprene Rubber (IR). (Linos, et. al, 2000).
Since 1914, natural rubber has been a classic subject of biodegradation studies.
(Rose and Steinbuchel, 2005). This is due to the high rate of its yearly manufacture which
is several million tons, as mentioned in the study of Bereeka (2006), and its slow rate of
natural degradation as reviewed by Rose and Steinbechul (2005). In fact, a number of
studies abound concerning its degradation. And it has been learned that both bacteria and
fungi can participate in such process. Throughout all the investigations and
experimentations done, two categories of rubber-degrading microbes according mainly on
growth characteristics have been established. Based on a review of Rose and Steinbuchel
(2005), which recapped the aforementioned groups, the microbes that can degrade rubber
can be categorized as clear zone-forming around their colonies and non-halo forming
whenever isolated and cultured in latex overlay plate, which is made by overlaying a layer
of latex agar medium on a basal salt medium agar. The former category was identified to
mainly consist of actinomycetes species. They are said to biodegrade or metabolize rubber
by secreting enzymes and other substances and also they are dubbed to be slow
degraders since they rarely show an abundant cell mass when grown on natural rubber
directly. On the other hand, members of the second group do not form halos on latex
overlay plates. They, unlike the first group, grow more when directly grown on natural
rubber. In a way, their growth on rubber could be described in an adhesive manner. The
second group is said to demonstrate a relatively stronger growth on rubber. Species
comprising this category are the Corynebacterium-Nocardia-Mycobacterium group. They
consist of the Gordonia polyisoprenivorans strains VH2 and Y2K, G. westfalica strain Kb1,
and Mycobacterium fortuitum strain NF4.
As demonstrated by a particular study by Linos, et al. (2000), the mechanisms that
the microbes undergo when biodegrading is colonization, biofilm formation and aldehyde
group formation after cultivating it on the surface of latex gloves. Such is revealed after
undergoing the Schiff reagent’s test. This is further examined under a scanning electron
microscope. In their methodology they have indicated that the preliminary screening
method to be used in finding potential rubber-degrading bacteria is by growing such
bacteria or microbe on the latex overlay or by latex film on the mineral agar plates. Growth
and colonization of the microbe in this medium would indicate its utilization of rubber as its
sole carbon source; hence, making it as a potential rubber-degrader.
Chicken Feathers
In the Philippines, chicken feathers aren’t a publicly recognized problem. But,
experiments and researches for its reuse and degradation are being explored at present. At
University of the Philippines-Los Baños, scientist Menandro Acda has ventured into
recycling chicken feather into a low-cost building material. The scientist quoted that,
recycling it would be more advisable than burning it since the incineration problem could
cause environmental hazards. (Morales, 2008). Moreover, in the US alone, 2 billion pounds
of chicken feathers are produced by the poultry industry (Comis, 2008). Chicken feathers,
by nature, are made up of over 90% protein (Cheng-cheng, et al., 2008). And this protein is
none other than keratin. It’s actually the most abundant protein. It is not easily degraded
due to its tightly packed structural arrangement which is in the form of alpha keratin or beta
keratin. The key to its stability lies on the cross-linking by disulfide bonds, hydrophobic
interactions, and hydrogen bonds. Such stability renders keratin water-insolube and non-
degradable by the enzymes papain, trypsin and pepsin. (Gradisar et al., 2005). In a study
conducted by Onifade, et al., 1998 and Goushterova et al., 2005, as cited in the journal of
Cheng-Cheng, et al., 2008, the build-up of chicken feathers in the environment and landfills
would only result to future pollution problems and protein wastage. More so, its
accumulation could serve as a breeding ground for a variety of harmful pathogens (Singh,
2004).
Considering that chicken feathers have a high protein content it could also be used
as an animal feed, but first its protein must be degraded (Tapia and Contiero, 2008). Yet
this is said to need so much water and energy (Frazer, 2004). Old methods of degrading
the chicken feathers such as alkali hydrolysis and steam pressure cooking are no longer
advisable. They cause so much energy wastage and they unfortunately destroy the
configuration of proteins. (Cheng-cheng, et al., 2008).
Incineration is also a method used in degrading such waste but it causes so much
energy loss and carbon dioxide build-up in the environment. Other methods of disposal are
landfilling, burning, natural gas production and treatment for animal feed. But subjecting it
to burning and land filing costs a lot and it contributes air, soil and water contamination.
(Joshi et al., 2007).
A wiser suggestion or approach would be the use of microbes in degrading these
chicken feathers. (Cheng-cheng, et al., 2008). Such approach is said to an economical and
environment-friendly alternative (Joshi, et al., 2007). Experiments that tested on the
degradation of chicken have already been done. In fact, studies have already proven that
keratinolytic microbes such as Bacillus (Maczinger, et al., 2003; Rodziewicz and Wojciech,
2008; Joshi et al., 2007), fungi (Gradisar, et al., 2005) and actinomycetes (Goushterova et
al., 2005). The enzymes that perform keratin degradation are called keratinase, which
could degrade feathers and make it available for its use as animal feed, fertilizer and
natural gas. The enzymes are said to degrade the beta-keratin component and the main
idea behind such biodegradation is that the microbes use the feather as their carbon,
nitrogen, sulfur and energy for their nourishment. (Joshi et al.,2007 ; Manczinger, et al.,
2003).
Keratinases isolated from microbes have various economic uses. Aside from its
feather degrading capacity, it could be used in the leather industry as an agent in dehairing
leather. Its by-product, the feather hydrolysate, could also be used as animal feed additive.
(Joshi, et al., 2007). Furthermore, potentially, the said hydolysate could be used in the
generation of organic fertilizer, edible films and amino acids which are considered rare, as
cited by Brandelli in the journal of Joshi et al. (2007).
In terms of experimental procedures, various methods are used in determining the
keratinolytic ability, which means it could produce keratinase and hence degrade chicken
feather, of microbes. A particular study by Tapia and Contiero (2008), used a feather meal
agar, wherein the feather served as the source of carbon, nitrogen, sulfur and energy, in
cultivating the isolated microbe Streptomyces. The growth, which occurred on the 10 th day
of incubation, through colony formation of the microbe indicated that it utilized the feather
as a source of its nutrients. After which, its keratinolytic activity was tested using a modified
keratin azure protocol. Another study by Maczinger et al. (2003) focused on the isolation of
a microbe from the poultry waste that could degrade feathers. During the preliminary
elimination, they cultured the different population of bacteria found in a partially degraded
feather in a basal medium with sterilized feathers serving as its source of carbon, nitrogen
and sulfur. It was then rotated in an orbital shaker for 10 days. After 4 days, one flask which
showed a visual degradation of the feather. A dilution series was made afterwards so as to
isolate and culture the bacteria that just degraded the feather. The strain was identified as
Bacillus lichenformis strain K-508. And the confirmation of the keratinolytic activity was
done by using the azokeratin as a substrate assay.
Isolation of a new microbial organism that could degrade chicken feather will help in
the degradation of the chicken feathers which is now becoming a burden in the society both
internationally and locally. The microbe could potentially provide the keratinase that could
be used in compost technology (Maczinger et al., 2003) or in the conversion of feather to
feedstock meal additives (Tapia and Contiero, 2008).
METHODOLOGY
I. Research Design
The research design to be used in the study is the Randomized Complete Block
Design (RCBD). The experiment will consist of two trials/runs with three replicates per
treatment. The experimentation process will be conducted in UP Los Baños Institute of
Biotechnology and at the Microbiology thesis room of the CAS, UP-Manila.
II. Experimentation
A. Preparation of Inoculum
The stock culture of Xylaria sp. and its four albino mutant strains will be obtained
from UPLB Biotech. Xylaria sp. will be isolated by culturing it in a Potato Dextrose Agar
(PDA) medium. The pH will be adjusted to pH 5 and it will be incubated at 25˚C. After 2-3
days, the fungi will be transferred into the mineral medium flask for enrichment and
sustenance to further growth.
B. Preparation of Pollutants
A. Polystyrene
1x1 cm strips will be cut from clean polystyrene food containers such
as coffee foam cups. The strips will be surfaced sterilized using 70% ethanol
solution. The strips will be weighed in three’s. The weight will serve as the
initial weight. Then the three strips that were weighed will be placed in a flask.
So, there will be three strips per replicate of each treatment.
B. Chicken feathers
Fresh feathers from Gallus sp. will be obtained from a nearby market
place where chickens are butchered and sold. The feathers will be cut into 3
cm in length. Each cut feather will be weighed and placed in a foil. The weight
obtained will serve as the initial weight. The feathers will be autoclaved for 20
minutes at 15psi. Then the 3 cm feather per replicate of each treatment will
be used and placed in a flask.
C. Rubber
Obtain rubber latex gloves size 5. Cut the gloves into strips of the
same sizes, approximate area to be about 2x2. Weigh the gloves by two’s.
The weight will serve as the initial weight. Then surface sterilize the gloves
using 70% ethanol. Use 2 strips per replicate of each treatment.
C. Biodegradation Proper using Culture Method
Two sets of flasks will be prepared containing 50 ml Mineral Medium each, in
triplicate. 0.5% glucose will be added in set A and B. The pH will be adjusted to pH 5 by
adding small amounts of either 0.1M NaOH or 0.1M HCl. For sterilizing purposes, the
media prepared will be exposed to UV light for 10 minutes. Then 2 ml of the microorganism
which came from the mineral medium flask will be added. The addition of pollutant in set B
only will follow right after. The flasks will be subjected in a shaker for four hours to
homogenize the medium. The culture will be observed for some time then when all the
glucose has been used up and the fungi have grown into a considerable mass as examined
visually, another MM + 0.5% glucose will be added in set A only, leaving the set B flasks to
utilize the solid pollutants as the sole carbon source. The extent of colonization will be
carefully examined every day until rate of colony growth can be predicted (growth in
mm/day). But if otherwise, the addition the MMG (mineral medium+0.5%glucose) to both
sets of flasks will be continued until the fungi have grown and thrived. Incubate for30 days,
with the flasks in a room with more or less 25 0C in temperature. The solid pollutants will
then be removed from the culture medium and examined under a scanning electron
microscope (SEM).
Note:
* This step is intended for each mutant and for the wild type. Since we have 4 mutant
strains and a wild type, this step will be repeated five times multiplied with the number of
pollutants to be used.
D. Determination of Amount of Degradation through Colonization
Incubation periods and set-up observations are done after 20, 30 and 50 days. After
each incubation period, the remaining pollutant will be weighed in grams. This
measurement will be recorded as the final weight. The percent weight loss of pollutant will
be determined using the formula:
% weight loss = (initial weight – final weight)
Initial weight
Statistical Analysis
The analysis that will be used for this study is the ANOVA for Random Complete
Block Design (RCBD). The blocks will be the Xylaria sp. and the mutants while the
treatment will be the three pollutants namely natural rubber, chicken feather and
polystyrene.
Hypothesis
(Hypotheses for the blocksF for rows)
Ho1: There is no significant difference in the biodegrading ability of the different Xylaria sp.
strains on the pollutants.
Ha1: There is a significant difference in the biodegrading ability of the different Xylaria sp.
strains on the pollutants.
(Hypotheses for the pollutantsF for columns)
Ho2: There is no significant difference in the degree of biodegradation of polystyrene,
natural rubber & chicken feathers due to Xylaria sp.
Ha2: There is a significant difference in the degree of biodegradation of polystyrene, natural
rubber & chicken feathers due to Xylaria sp.
Dummy Tables
Table 1. Percent Weight Loss in two runs of the (insert pollutant name here) due to
Xylaria sp. strains
Replicate Replicate Replicate Replicate Replicate Replicate Mean
1 2 3 4 5 6
Wild
type
Mutant
1
Mutant
2
Mutant
3
Mutant
4
Note:
*replicate 1-3 = run 1
**replicate 4-6 = run 2
***Table 1 will be used for each pollutant hence it will be repeated thrice for each of the
three incubation periods.
Table 2. Mean Values of the Percent Weight Loss of Polystyrene, Natural Rubber and
Chicken Feathers due to degradation of Xylaria sp. strains
Xylaria strain Polystyrene Natural Chicken
Rubber Feathers
Wild-type
Mutant1
Mutant2
Mutant3
Mutant4
Table 3. ANOVA for Randomized Complete Block Design of the Potential
Biodegrading Ability of Xylaria sp. Strains on Chicken Feathers, Natural Rubber and
Polystyrene
Source of Variation SS df MS F P-value F crit
Rows (Xylaria
strain)
Columns (pollutant)
Error
α = 0.05
In the event that the F value for rows or columns shows any significant difference,
a post hoc analysis would be conducted. For the F value for rows, which are the pollutants,
the Multiple Analysis test will be used and it will be followed by the Tukey’s test. While for
the F value for columns, the Dunnet’s test will be applied.
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